We wish to continue our studies on the conformational properties of native and selectively modified hemoglobins in an effort to learn the mechanistic basis of the more critical regulatory processes carried out by the protein. We intend to probe further into part of the organic phosphate binding site in fetal hemoglobin by examining the pH dependency of the effects of organic phosphates on oxygen affinity and methemoglobin conformation in the fetal protein. Preliminary results indicate that fetal hemoglobin may bind organic phosphates as effectively as does the adult protein below pH 6.8; results of further studies would have strong implications for the effects of acidosis in the pregnant woman on fetal respiration. Additional results suggest that organic phosphates may reduce the solubility of methemoglobin S, and raise the possibility of complications of methemoglobinemia in the sickle cell patient. We hope to examine the region, near the amino terminus of the beta chains, responsible for the unique properties of hemoglobin S, by testing for aggregation, in the met- protein in the presence of organic phosphates, and in enzymatically modified and chemically deaminated deoxy- proteins. We plan to investigate the role of the amino-terminal residues in normal hemoglobin function by specific deamination with phenylglyoxal. With this approach we can probe the role of the amino termini in organic phosphate binding, complexing with CO2, and contributing to part of the Bohr effect, without producing altered properties arising from the presence of an attached modifying reagent. Finally, we wish to complete our studies on the conformational equilibria in carboxypeptidase-digested hemoglobins and in methemoglobin in order to quantitate the relationship between the magnitude of functional cooperative interactions and the ratio of oxy- to deoxy-quarternary structures. Our assays of functional properties include oxygen equilibrium and Bohr effect determinations, ultracentrifugation, and measurement of rate of enzymatic digestion. Conformational probes include circular dichroism, sulfhydryl reactivity, and nuclear magnetic resonance.